PB1711: DROPLET DIGITAL PCR DETECTION OF THE T315I BCR::ABL1 KD MUTATION IN ADULT PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: Deborah Cardinali, Marco Beldinanzi, Michela Ansuinelli, Loredana Elia, Irene Della Starza, Vittorio Bellomarino, Mabel Matarazzo, Mariangela DI Trani, Mattia Cola, Anna Rita Guarini, Robin Foà, Sabina Chiaretti
Journal: HemaSphere (2023)

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is characterized by the reciprocal translocation t(9;22)(q34;q11). The advent of tyrosine kinase inhibitors (TKIs) has markedly improved the outcome of Ph+ ALL patients and indeed changed the natural history of the disease. At present, TKIs represent the gold standard treatment for patients with Ph+ ALL, with or without chemotherapy1. Despite the efficacy of targeted therapy with TKIs, some patients show drug resistance due to the onset of BCR::ABL1 kinase domain (KD) point mutations. In particular, the most deleterious one is represented by the T315I, which cause treatment failure with first and second generation TKI-based therapies. Sanger sequencing (SS) is the gold standard tool for BCR::ABL1 KD mutational screening. The digital droplet PCR (ddPCR) technology, a third generation PCR, may represent a valid alternative to SS for the detection of BCR::ABL1 KD mutations. Aims: The aim of this study was to evaluate if ddPCR is at least as sensitive as SS for the detection of the T315I mutation, and if the ddPCR can detect the mutation also at very low levels of minimal residual disease (MRD), ultimately anticipating molecular relapse. Methods: The study comprised samples from patients enrolled in the phase II GIMEMA LAL2116 chemotherapy-free protocol for newly diagnosed adult Ph+ ALL and based on the administration of the second generation TKI dasatinib followed by the bispecific monoclonal antibody blinatumomab2. A BCR::ABL1 KD mutational screening was carried out in all patients with a MRD increase by both SS and ddPCR. In mutation-positive patients, the time-point (TP) preceding the MRD increase was evaluated by ddPCR to assess the earlier presence of the mutation. DdPCR was performed as described3. Results: We carried out a BCR::ABL1 KD mutational screening by SS in 16 patients enrolled in the GIMEMA LAL2116 protocol. Overall, of the 16 patients with a MRD increase (10 during the induction phase, 6 in the consolidation phase), 8 resulted wild type (WT) while mutations were detected in 8 patients (7 harboring the T315I mutation and 1 the E255K). In the T315I positive patients, the evaluation by ddPCR of the same TP samples, confirmed in all cases the presence of the mutation. More importantly, the analysis at a previous TP, when MRD levels were lower, showed that ddPCR could detect the T315I BCR::ABL1 KD mutation in 6/7 cases, while 1 positive non-quantifiable (PNQ) case proved WT (Table 1). In order to determine if ddPCR could anticipate the detection of mutations compared to SS, the previous TPs were also evaluated by SS in 4 cases with available material. In 2 cases the mutation was detected also by SS, while in 2 it could not be found (Table 1). Likewise, ddPCR for the T315I mutation was also performed in samples that proved negative by SS, and the absence of mutations was confirmed; this is particularly relevant from a clinical standpoint and was corroborated by the fact that only 1/8 experienced a central nervous system relapse in this last cohort, and is currently alive in second complete remission (CR). Summary/Conclusion: The ddPCR proved as reliable and accurate as SS to detect the T315I BCR::ABL1 KD mutation. Furthermore, the ddPCR proved to be more sensitive for predict molecular relapse before the increase in MRD where, in some cases, the SS failed to detect the T315I mutation. Further efforts are ongoing to expand this screening also to other ABL1 mutations, considering the always more frequent use of ponatinib in the first-line setting. 1. Foà & Chiaretti, NEJM 2022 2. Foà et al, NEJM 2020 3. Soverini et al, Leukemia 2022Keywords: Acute lymphoblastic leukemia, BCR::ABL, Droplet Digital PCR (ddPCR), Mutation analysis